Maize (Zea mays L.) is the third largest planted crop after wheat and rice in Iran. Unfortunately, this crop is vulnerable to diverse fungal species attack and mycotoxin contamination in field and storage. Species belonging to Fusarium genus, are among the most important fungal pathogens of maize and affect its productivity and kernel quality. In this research, in order to accurately identify of 234 strains of Fusarium isolated from maize in Iran, we used sequences of EF-1α gene. Based on morphological characteristics, several different species were identified, namely F. verticillioides, F. proliferatum, F. subglutinans, F. thapsinum, F.redolens, F. solani, F.sacchari and species belonging to F. incarnatum equiseti species complex. However, the EF-1α gene sequences allowed us to identify a wider number of species: F. verticillioides, F. proliferatum, F. subglutinans, F. thapsinum, F. temperatum, F.nygamai, F.redolens, F. brachygibbosum, F. solani and FIESC speecies. Based on EF-1α gene sequences, the 21 strains belonging to the FIESC complex were divided into 7 separate phylogenetic groups. Also, all isolates described as F. sacchari, based on morphological characteristics, were identified as F. subglutinans through molecular analyses. All these species are able to produce a wide range of mycotoxins which are harmful to human and animals. Moreover, each species has its own mycotoxin profile. Therefore, the occurrence of such high level of different species on Iranian maize could be reason of great concern because of the toxigenic risk associated to these species. Finally, is worthwhile to underline that this is the first report of the presence of F. temperatum and F. brachygibbosum in maize grains cultivated in Iran.